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Patulin and citrinin are mycotoxins produced by certain fungi mainly belonging to Penicillium and Aspergillus and may be detectable in mouldy fruits and fruit products. The data presented in this study refer the simultaneous occurrence of patulin and citrinin in 351 samples of seven different varieties of apples with small rotten areas (Casanova, Golden Delicious, Red Delicious, Reineta, Richared, Rome Beauty and Starking). It used a rapid multi-detection thin layer chromatography (TLC) method. The minimum detectable concentrations of the patulin and the citrinin were 120-130 micrograms/kg and 15-20 micrograms/kg respectively. The percentage of contamination with patulin only was higher ( 68.6 %) than the percentage of contamination with citrinin only (3.9 %). Patulin and citrinin (19.6%) were also detected simultaneously. The highest mean patulin content was 80.50 mg/kg for the Richared variety, but the mean level of citrinin was lower. The lowest mean contaminations of patulin were found in Rome Beauty, Red Delicious and Reineta, ranging from 3.06 to 5.37 mg/kg. All analyzed apples varieties had low citrinin contamination, ranging from 0.32 to 0.92 mg/kg. These findings indicate that there may be a risk of human exposure to patulin through the consumption of juices and jams manufactured with apples with small rotten areas.

Key words : Patulin, citrinin, TLC, co-occurrence, apples.



Introduction

Patulin and citrinin are toxins produced by moulds of the genera Aspergillus and Penicillium (Steiman et al. 1989), including A. clavatus, P. patulum and P. expansum (Scott 1994). They can be detected in mouldy fruit, such as apples, pears (Harwig et al. 1973, Scott 1974, Ciegler et al. 1977, Frank 1977, Burdaspal and Pinilla 1979, Mortimer et al. 1985, Martins 1987), and in cereals (Harwig et al. 1977, Lopez-Diaz and Flannigan 1997). Patulin is particularly associated with brown rot in apples (Krogh 1987). The minimum aw value for patulin production are 0.99 and 0.95 and the temperature range were 0 to 24ºC and 4 to 31ºC for P. expansum and P. patulum, respectively (Northolt et al. 1978).

Patulin (4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one) is a toxic substance with suspected carcinogenic properties (McKinley et al. 1982). Pohland and Allen (1970) reported that patulin rapidly reacts with sulfur dioxide (SO2) so that it could be destroyed when the latter is used as an antioxidant or antimicrobial agent. Its biological activity is decreased in an alkaline medium and in presence of molecules comprising sulfhydryl group such as cysteine and glutathione (Lindroth 1980). From experiments it has been demonstrated that while the patulin is eliminated during fermentation in wines (Ough and Corison 1980), its content is decreased only by about 20% during the usual technological process of fruit juice production (Harrison 1989). Bissessur et al. 2001 showed that patulin was significantly reduced in apple juice when clarification procedures were employed. Therefore, pressing followed by centrifugation resulted in an average toxin reduction of 89%. The total toxin reduction using filtration, enzyme treatment and fining were 70, 73 and 77%, respectively. Clarification was successful in the reduction of patulin in apple juice. Nevertheless, clarification resulted in high levels of patulin in the pressed pulp after filtration and centrifugation, and this could be harmful if they were used as animal feeds (Bissessur et al. 2001). Patulin was originally described as an antibiotic and exhibits strong antibiotic activity against different Gram-positive and Gram-negative bacteria including Mycobacterium tuberculosis. It also possesses antifungal activity and it is highly toxic to plant and animal cells in tissues (Sorenson et al. 1985). Several studies indicate the immunosuppressive action of patulin, which is known for producing ulceration, congestion, and haemorrhagic lesions, particularly in the gastrointestinal tract (McKinley et al. 1982).

Combined reproductive toxicity, long-term toxicity/carcinogenicity studies in rats have established a provisional maximum tolerable daily intake (PMTDI) of 0.4 micrograms/kg bw , based on a no-effect level (NOEL) of 43 micrograms/kg bw/day ( JECFA,1995).

Citrinin (3R-trans)-4,6-dihydro-8-hydroxy-3,4,5-trimethyl-6-oxo-3H-2-benzo-pyran-7-carboxylic acid (Cole and Cox 1981) was first identified as a secondary metabolite of P. citrinum, from which it derived its name. The site of effects of citrinin is primarily the kidneys and liver; within a few hours after citrinin administration, DNA, protein, and glutathione (GSH) content in these tissues are decreased (Cheeke and Shull 1985). Campbell et al. (1981) demonstrated that citrinin appears to be immunostimulatory rather than immunosuppressive; however, it remains to be determined whether ingestion of feed contaminated with low levels of citrinin produces responses similar to intraperitoneal exposure. Citrinin has been suggested as a causative factor in renal disease among livestock, poultry, pigs, rats and dogs (Reiss 1977, Cheeke and Shull 1995,). Citrinin was strongly implicated as the cause of pruritis, pyrexia and haemorragic syndrome in cattle (Griffiths and Done 1991). The renal system of humans is affected the most and the mitochondrial respiratory chain was identified as a possible sensitive target for this mycotoxin (Ammar et al 2000). It is commonly found in grain samples along with ochratoxin A and these two mycotoxins have been suggested as the cause of porcine nephropathy in Denmark (Betina 1989). Citrinin is a primarily known as nephrotoxin, and few studies have addressed its potential for immunotoxicity (Sharma 1993).
Several countries have set limits for the content of patulin in foods of 50 micrograms/kg or 50 micrograms/L (FAO 1997). There is no specific legislation for citrinin; nevertheless, the FAO (1997) refers to a level of zero for ´all mycotoxins` in preserved food in Hungary, legume products in The Netherlands and ice cream in Trinidad.

The aim here was to provide information on the occurrence and levels of patulin and citrinin in different varieties of apples with small brown rotten areas in a significant sampling. The ratio of weight of the rotten area to the whole apple was about 1/3. We determined these mycotoxins using a rapid multidetection TLC method (Gimeno and Martins 1983, Gimeno 1984)



Results and Discussion

A total of 351 apples with different-sized brown rotten areas and of different varieties (44 Casanova, 60 Golden Delicious, 28 Red Delicious, 62 Reineta, 40 Richared, 42 Rome Beauty and 75 Starking) were collected randomly from markets and supermarkets in Portugal for determination of patulin and citrinin by a rapid multi-detection TLC method. The whole apple and the respective rotten area were weighed and it was found that from 351 samples the rotten area represented an average of 32 % of the whole apple; they ranged from 25 to 42 % (SD = 22). There was no correlation between the concentration of patulin and/or citrinin and the percentage of the rotten area in the whole apple, e.g. apples with a lower percentage of rotten area than the others showed a significantly higher mycotoxin concentration than the others, and apples with a higher percentage of rotten area than the others showed a significantly lower mycotoxin concentration than the others.

A total of 241 samples of the 351 analyzed (68.6 %) contained patulin only (table1). Fourteen samples (3.9%) were contaminated with citrinin only and 69 samples (19.6%) were simultaneously contaminated with patulin and citrinin. Twenty-seven (7.6%) were negatives. (table 1). All apples analyzed had a higher incidence of patulin contamination compared with that of citrinin; and of the seven analyzed varieties, the Red Delicious and Reineta showed greater frequency of contamination (table 1).

The highest mean patulin content was 80.5 mg/kg for the Richared variety; nevertheless the citrinin contamination was the lowest (0.32 mg/kg) (table 2). Casanova and Starking varieties had mean patulin concentrations of 33.10 and 13.67 mg/kg; Reineta, Red Delicious, Rome Beauty and Golden Delicious showed low contamination, ranging from 5.37 to 3.05 mg/kg. (table 2).

All analyzed apples varieties had lower citrinin contamination compared with patulin contamination. Rome Beauty variety had the highest mean level of citrinin (0.92 mg/kg) but a low mean level of patulin. The others varieties had mean citrinin levels ranging from 0.32 to 0.76 mg/kg. (table 2).

In this study we also verified that the patulin had spread to the areas not affected by rot. We did not found citrinin in these areas, and this may be explained by the lower content of this mycotoxin in apples analyzed.

Surveillance studies of patulin in apples and apple-based food were reported by Beretta et al. (2000). Their data showed lower contamination with levels ranging from 0.71 to 1170 micrograms/kg. Patulin was also found by Frank (1977) in 50% of different varieties of apples and pears with brown-rotten areas (about 120 samples). Fritz et al. (1979) found patulin concentrations ranging from 0.002 to 0.3 mg/L in commercial apple juice and from 0.3 to 42 mg/kg in the brown-rotten portions of apples. In Brazil, in 30 test samples of apple juice, only one was found to contain patulin at 17 micrograms/L, (deSylos and Rodriguez-Amaya 1999). Viñas et al. (1993) studied the citrinin producing capacity of 122 Penicillium expansum strains isolated from apples from Lleida (Spain) and verified that among the strains examined 46% produced citrinin in a culture medium (glucose yeast agar[GYA]) and 73 % of these strains were isolated from decayed apples.

The mean levels of patulin and citrinin found in the present survey were higher than those presented by the others researchers mentioned above. This may be due to the superior toxigenic potential of the indigenous Penicillium strains, to the substrate composition or to the physical ecological conditions (temperature, pH ). The values show that if apple juice and apple-based foods are prepared with low-quality fruit the presence of patulin and citrinin can be higher than the safe limits established by international committees and this could be true even when the brown rotten area is removed from the apples. According to FAO (1997), limits of 50 micrograms/L of patulin have been set for fruit derivatives and the data presented in this study indicate mean concentration levels above this. According to the same reference (FAO 1997) there are no specific levels established for citrinin in foods or feeds.




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